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IMMUNOTHERAPY

GM-CSF disruption in CART cells modulates T cell activation and enhances CART cell anti-tumor activity

Leukemia volume 36, pages 1635–1645 (2022)Cite this article

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Abstract

Inhibitory myeloid cells and their cytokines play critical roles in limiting chimeric antigen receptor T (CART) cell therapy by contributing to the development of toxicities and resistance following infusion. We have previously shown that neutralization of granulocyte-macrophage colony-stimulating factor (GM-CSF) prevents these toxicities and enhances CART cell functions by inhibiting myeloid cell activation. In this report, we study the direct impact of GM-CSF disruption during the production of CD19-directed CART cells on their effector functions, independent of GM-CSF modulation of myeloid cells. In this study, we show that antigen-specific activation of GM-CSFKO CART19 cells consistently displayed reduced early activation, enhanced proliferation, and improved anti-tumor activity in a xenograft model for relapsed B cell malignancies. Activated CART19 cells significantly upregulate GM-CSF receptors. However, the interaction between GM-CSF and its upregulated receptors on CART cells was not the predominant mechanism of this activation phenotype. GM-CSFKO CART19 cell had reduced BH3 interacting-domain death agonist (Bid), suggesting an interaction between GM-CSF and intrinsic apoptosis pathways. In conclusion, our study demonstrates that CRISPR/Cas9-mediated GM-CSF knockout in CART cells directly ameliorates CART cell early activation and enhances anti-tumor activity in preclinical models.

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Fig. 1: GM-CSF directly contributes to CART cell activation and apoptosis.
Fig. 2: GM-CSF knockout via CRISPR/Cas9 does not impair CART19 production and effector functions.
Fig. 3: GM-CSF disruption on CART19 modulates early activation and anti-tumor activity.
Fig. 4: Activated T cells and CART19 cells express high levels of GM-CSF receptors.
Fig. 5: GM-CSFKO CART19 are intrinsically more resistant to apoptosis.

Data availability

Sequencing data are available at BioProject PRJNA623000.

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Acknowledgements

Mayo Clinic Cancer Center (SK).

Funding

Exact Foundation (SSK), Humanigen (SSK), Mayo Clinic Center for Clinical and Translational Science grant UL1TR002377 (SSK), Mayo Clinic Center for Individualized Medicine (SSK), National Comprehensive Cancer Network (SSK), National Institutes of Health grants K12CA090628 (SSK) and R37CA266344-01 (SSK), Department of Defense grant CA201127 (SSK), and Predolin Foundation (RS and SSK).

Author information

Author notes

  1. These authors contributed equally: Michelle J. Cox, Claudia Manriquez Roman, Reona Sakemura.

Authors and Affiliations

  1. T Cell Engineering, Mayo Clinic, Rochester, MN, USA

    Michelle J. Cox, Claudia Manriquez Roman, Erin E. Tapper, Elizabeth L. Siegler, Mehrdad Hefazi, Kendall J. Schick, Paulina Horvei, Michael W. Ruff, Ismail Can, Mohamad Adada, Evandro Bezerra, Lionel Aurelien Kankeu Fonkoua, Reona Sakemura & Saad S. Kenderian

  2. Division of Hematology, Mayo Clinic, Rochester, MN, USA

    Michelle J. Cox, Claudia Manriquez Roman, Erin E. Tapper, Elizabeth L. Siegler, Sutapa Sinha, Mehrdad Hefazi, Kendall J. Schick, Ismail Can, Mohamad Adada, Evandro Bezerra, Lionel Aurelien Kankeu Fonkoua, Sameer A. Parikh, Neil E. Kay, Reona Sakemura & Saad S. Kenderian

  3. Bioinformatics and Computational Biology, University of Minnesota Graduate School, Minneapolis, MN, USA

    Michelle J. Cox, Raphael Mwangi & Nancy S. Scott

  4. Department of Molecular Medicine, Mayo Clinic, Rochester, MN, USA

    Claudia Manriquez Roman & Saad S. Kenderian

  5. Mayo Clinic Graduate School of Biomedical Sciences, Rochester, MN, USA

    Claudia Manriquez Roman, Kendall J. Schick, Ismail Can & Saad S. Kenderian

  6. Humanigen, Inc, Burlingame, CA, USA

    Dale Chappell, Cameron Durrant & Omar Ahmed

  7. Biomedical Statistics and Informatics, Mayo Clinic, Rochester, MN, USA

    Raphael Mwangi

  8. Center for Individualized Medicine, Mayo Clinic, Rochester, MN, USA

    Nancy S. Scott

  9. Department of Molecular Pharmacology & Experimental Therapeutics, Mayo Clinic, Rochester, MN, USA

    Kendall J. Schick

  10. Department of Pediatric Hematology/Oncology, Mayo Clinic, Rochester, MN, USA

    Paulina Horvei

  11. Department of Neurology, Mayo Clinic, Rochester, MN, USA

    Michael W. Ruff

  12. Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA

    Ismail Can

  13. Department of Oncology, Mayo Clinic, Rochester, MN, USA

    Mohamad Adada, Evandro Bezerra & Lionel Aurelien Kankeu Fonkoua

  14. Department of Immunology, Mayo Clinic, Rochester, MN, USA

    Saad S. Kenderian

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  1. Michelle J. Cox
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Contributions

SSK formulated the initial idea; MJC, CMR, RS, and SSK designed experiments; MJC, CMR, RS, EET, ELS, and SS performed experiments; MJC, CMR, RS, and SS analyzed data; MJC, RM, and NS analyzed sequencing data; SAP and NEK contributed reagents; SSK supervised the study. All authors edited and approved the final version of the manuscript.

Corresponding author

Correspondence to Saad S. Kenderian.

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Competing interests

SSK is an inventor on patents in the field of CAR immunotherapy that are licensed to Novartis (through an agreement between Mayo Clinic, University of Pennsylvania, and Novartis). MJC, RS, RMS and SSK are inventors on patents in the field of CAR immunotherapy that are licensed to Humanigen (through Mayo Clinic). MH and SSK are inventors on patents in the field of CAR immunotherapy that are licensed to Mettaforge (through Mayo Clinic). SSK receives research funding from Kite, Gilead, Juno, BMS, Novartis, Humanigen, MorphoSys, Tolero, Sunesis/Viracta, Leahlabs, and Lentigen. NEK receives research funding from Acerta Pharma, BMS, Pharmacyclics, MEI Pharma, and Sunesis. NEK has participated in Advisory Board meetings of Cytomx Therapy, Janssen, Juno Therapeutics, Astra Zeneca, and Oncotracker; and on DSMC for Agios and Cytomx Therapeutics. SAP receives research funding from Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, BMS, AbbVie, and Ascentage Pharma. SAP has participated in Advisory Board meetings of Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie (he was not personally compensated for his participation). CD, DC and OA are employees of Humanigen.

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Cox, M.J., Manriquez Roman, C., Tapper, E.E. et al. GM-CSF disruption in CART cells modulates T cell activation and enhances CART cell anti-tumor activity. Leukemia 36, 1635–1645 (2022). https://doi.org/10.1038/s41375-022-01572-7

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